The mating type locus (MAT) encodes several DNA-binding proteins, which determine the cell types of Saccharomyces cerevisiae: the a and alpha haploid cell types, and the a/alpha diploid cell type. Three of the four MAT transcriptional units contain homeodomains, regions which show homology to the developmentally important homeotic genes of multicellular eukaryotes. MATalpha2 encodes a repressor that regulates two different sets of genes. As a homodimer alpha2 binds to an operator at the a- specific genes. In a/alpha diploid cells alpha2 interacts with a1, a product of the other MAT allele, to repress the haploid-specific genes. The long term goal of this research is to understand the mechanism by which MATa1 determine the diploid cell type in yeast. Using in vitro translated proteins we demonstrated that a1 is a DNA binding protein that forms a heterodimer with alpha2 which binds to an operator at the haploid-specific genes. We have recently tested a complete set of mutant operators for binding to a1/alpha2 and have found two clusters of bases whose sequence is important for binding. We propose to further characterize the molecular interactions of a1 by performing studies with the following specific aims: 1. To determine the orientation of the a1/alpha2 heterodimer on its operator by per\forming DNA-protein crosslinking experiments with BrdU-substituted radiolabelled operators. 2. To identify regions of a1 protein that are in close proximity to the operator DNA by purifying the photo-crosslinking peptide-DNA complexes and sequencing the peptides. 3. To create a set of random mutations in MATa1 by in vitro mutagenesis, that can be screened to identify domains responsible for DNA binding, repression of transcription, and protein- protein contacts with alpha2 and other proteins required for repression. Mutations will be screened in vivo their ability to repress a haploid- specific gene, STA1, that encodes an amylase whose secretion will be assayed using a convenient colony halo assay on starch plates. The mutant a1 genes will be used as templates for in vitro synthesis of the protein to test DNA-binding and heterodimer formation in vitro. 4. To establish which side chains of the protein are responsible for base sequence recognition by selecting mutant a1 proteins that recognize altered DNA sequences. The mutant a1 sequences generated in Specific Aim 3 will be tested against our set of mutant operators. The operators will be cloned into a vector expressing URA3. Mutant a1 proteins that repress transcription at the mutant operators will be selected using 5-fluoro- orotic acid, which selects against cell expressing URA3. The sequence of mutant MATa1 genes with altered DNA recognition properties will define the side chains responsible for sequence recognition.